To understand the molecular mechanisms underlying neurotransmitter release and its modulation, we are identifying proteins that control the trafficking and targeting of presynaptic release machinery to nerve terminals, regulate SNARE complex assembly and diassembly, and modulate synaptic vesicle recycling. Syntaphilin: an inhibitory modulator of both SNARE-dependent fusion and dynamin-mediated endocytosis. Synaptic vesicle exo- and endocytotic processes must be effectively coupled so as to maintain a level of synaptic homeostasis compatible with neurotransmission. One mechanism by which these criteria could be met would require presynaptic proteins that could modulate the dynamics of neurotransmission by directly interacting with the basic machinery of synaptic vesicle exocytosis and endocytosis. Syntaphilin is a neuronal protein that we first characterized as a binding partner of syntaxin-1. Binding of syntaphilin to syntaxin inhibits the binding of syntaxin to SNAP-25 and thus prevents the formation of the SNARE core complex. Functionally, overexpression of syntaphilin in cultured hippocampal neurons inhibits neurotransmitter release; furthermore, injection of the syntaphilin syntaxin-binding peptide into the presynaptic cell body of superior cervical ganglion neurons results in inhibition of neurotransmission. As calcium influx into nerve terminal signals the onset of both synaptic vesicle exocytosis and compensatory endocytosis, we asked whether an analogous inhibitory clamp of the endocytosis protein complex might be present in synapses in which syntaphilin is expressed. We have demonstrated that, in addition to its effects on the synaptic vesicle release machinery, syntaphilin binds to dynamin-1 and inhibits its interaction with amphiphysin, consequently decreases clathrin-dependent, dynamin-mediated transferrin uptake in COS cells and synaptic vesicle recycling in cultured hippocampal neurons. Our findings implicate syntaphilin as an inhibitory modulator of both SNARE-driven exocytosis and dynamin-mediated endocytosis, and suggest a two-fold mechanism by which syntaphilin inhibits neurotransmitter release in neurons. Ca2+-dependent phosphorylation of syntaxin-1A by DAPK. By using yeast two-hybrid screening with syntaxin-1A as bait, we have isolated a cDNA encoding DAPK. Expression of DAPK in the adult rat brain is restricted to particular neuronal subpopulations, including the hippocampus and cerebral cortex. Biochemical studies demonstrate that DAPK binds to and phosphorylates syntaxin-1A at serine-188. This phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner. Syntaxin-1A mutation S188D, which mimics a state of complete phosphorylation, significantly decreases syntaxin binding to Munc18-1, a syntaxin-binding protein that regulates SNARE complex formation. Our results suggest that syntaxin is a DAPK substrate, and delineate a novel signal transduction pathway by which syntaxin function is regulated in response to intracellular [Ca2+] and synaptic activity. In addition, our finding that DAPK is enriched in synapses opens a new avenue of study to examine the cellular signal mechanisms by which the efficacy of neurotransmission is modulated in response to synaptic activity. Syntabulin: a novel syntaxin-binding and microtubule-associated protein implicated in syntaxin-cargo trafficking in neurons. Different types of organelles including synaptic vesicle precursors and cargo vesicles containing synaptic plasma membrane proteins such as syntaxin are sorted and transported from cell body to nerve terminal along the microtubules and participate in active zone formation by fusion with the presynaptic plasma membrane. Linking of cargo vesicles with their transport motors must be functional specific to preserve organelle identity and the proper flow of cargoes within the cell. However, the relationship between these cargoes and linker molecules connecting with their microtubule-based motors is unclear. In an effort to characterize syntaphilin isoforms, we have identified a brain-specific gene encoding a novel syntaxin-binding protein named syntabulin. Biochemical and immunocytochemical analysis showed that syntabulin is associated with microtubules and through this association attaches syntaxin-containing cargoes to microtubules. Our syntabulin loss-of-function studies with syntabulin SiRNA and overexpression of the syntaxin-binding peptide demonstrated that knockdown of syntabulin expression and the interference of syntabulin function significantly block attachment of syntaxin-cargo vesicles to microtubules and consequently reduce syntaxin distribution in the processes of cultured hippocampal neurons. Our studies suggest that syntabulin acts as a linker molecule capable of attaching syntaxin-cargo vesicles to an unknown molecular motor along microtubules and transporting them to neuronal processes, thus providing insight into the cellular mechanisms underlying the trafficking of presynaptic protein in neurons. We believe that our continued application of these approaches, in combination with a greater focus on siRNA knock-down and germ-cell genetic and functional analysis, will allow us to better understand the regulatory pathways that maintain and modulate presynaptic function.